Hexestrol Diazirine Photoaffinity Labeling Reagent for the Estrogen Receptor

3-Azibutyl (2R*,3S*)-2,3-bis(4-hydroxyphenyl)pentyl sulfide (1), a photoaffinity labeling reagent for the estrogen receptor (ER), has been prepared in unlabeled and in high specific activity tritium-labeled form (32 Ci/mmol) and has been shown to undergo selective and efficient photocovalent attachment to rat uterine ER. Diazirine 1 demonstrates high binding affinity for ER, as determined by both a competitive binding assay and a direct binding assay (relative binding: estradiol = 100; (1) = 17. K_d: estradiol = 0.19 nM; (1) = 0.98 nM, respectively). It is efficient in site-specific photoinactivation of ER, reaching the level of 31% after 5 min of irradiation at >315 nm. The tritium-labeled diazirine [³H]-1 undergoes specific photocovalent attachment to ER with an attachment efficiency of 29% and a selectivity of 90 %. Both of these values are quite high for a photoaffinity reagent. SDS—polyacrylamide gel electrophoretic analysis of the photolabeled proteins shows specific labeling of a major species at M_r 65 000, the same species that is labeled by [³H]tamoxifen aziridine, a well-characterized affinity label for ER. Hexestrol diazirine 1 is the first carbene-generating photoaffinity label that covalently labels ER with high efficiency and selectivity, and it should be useful in further studies on the hormone-binding domain of ER.